This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Folding of globular proteins involves the burial of hydrophobic sidechains. Thus, a series of dehydration events are expected to occur during the course of folding, especially in the formation of the hydrophobic core. For example, the search towards the native fold of the SH3 domain has been shown to involve an obligatory desolvation/water exclusion step occurring late in the respective folding process. Similarly, a dehydration step following the formation of the transition state was observed in the folding simulations of protein A. Despite the apparent importance of hydration/dehydration events in protein folding/unfolding, it has been difficult to tease out such processes from thermally or chaotropically induced protein denaturation transitions measured by the commonly used spectroscopic methods, even though the resulting signal of the spectroscopic probe thus employed depends on the degree of hydration. Here, we take advantage of the sensitivity of the CO stretching vibration to its immediate environment and use it as an infrared (IR) marker to probe the role of water in the unfolding transition of carbonmonoxy myoglobin (MbCO).